South Asian Journal of Experimental Biology, Vol 4, No 6 (2014)

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Jul 12
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In-vitro validation of self designed siRNA targeting non-structural 1 gene of Influenza A virus

Bhawana Jain, Amita Jain, Om Prakash, Ajay K. Singh, Tanushree Dangi, Mastan Singh, K. P. Singh

Abstract


 

The genomic variability makes Influenza A virus (IAV) difficult to be con-trolled by existing vaccines or anti-influenza drugs. Viral gene targeting siRNA induces the RNAi mechanism in the host and silents the gene by cleaving mRNA. Objective was to develop siRNA targeting non-structural 1 gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence align-ment) of NS1 gene of IAV strains. To choose the most efficient siRNA, three levels screening method was developed. Ultimately one siRNA duplex was selected on the basis of its unique position in conserved region. siRNA effica-cy was confirmed in vitro on commonly used Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/2011(H3N2)] and Influenza A/pdmH1N1 [A/India/LKO2151/2012(H1N1)]. Of total 173 strains worldwide and 30 strains from India, 32 bp long (position 561 - 592) conserved region was identified. The longest ORF of NS1 gene was targeted by the selected siRNA, which showed 65.5% inhibition in replication of Influenza A/pdmH1N1 and 67.2% inhibition in replication of Influenza A/H3N2 at 48 hpi on MDCK cell line. This study shows that siRNA targeting NS1 may be quite effective in controlling IAV rep-lication so can be used as anti-IAV therapeutic agent.


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